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Chapter 5

“SORTING OUT OF INTERFERENCE IN

DETECTION OF ENDOTOXINS IN

BIOTHERAPEUTIC DRUGS,

LACTOBIONIC ACID & MEDICAL DEVICES”

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5.1 Biotherapeutic drugs

Abstract:

The effect of varying interference factors responsible for false positive

results was investigated. The interfering factors inhibiting the Endotoxins in

activated lymphocytes sample was sorted by heat denaturation. These

activated lymphocytes are Biotherapeutic drugs which are used for

autologous immune enhancement therapy. The technique used for

denaturation can be applied to protein samples as well, to get rid of inhibition

while quantifying Endotoxins.

Introduction:

Water is the principal source of Endotoxin in parenteral products. In

general, as per the United State Pharmacopeias (USP) the threshold

pyrogenic dose is 5 EU/kg/hr for parenteral drugs and 0.2 EU/kg/hr for

intrathecal drugs. When Endotoxin enters into human blood these toxins

induces white blood cells (WBC) to release cytokines, such as Tissue Necrosis

Factor (TNF), Interleukin-1 and Interleukin-8, which mediate a complex

biological response including pyrogensity, shock, coagulation and

inflammation (Bang FB, 1956). So it is mandatory to check the presence of

Endotoxin level in Biothereaputic drugs before passing the product into

market.

Bacterial Endotoxin is one of the most potent activator of mammalian

immune system. Gram negative bacterial outer membrane

Lipopolysaccharide (LPS) induces a cascade of defense mechanism that is

known as fever and inflammation (C.M.Good and H.E. Lane, 1977). In

incomparable fashion, for parenteral drugs administered into cerebrospinal

fluid (CSF) known as intrathecal administration, K is reduced to 0.2 EU/kg. The

most common toxic route of entry of Endotoxin into human system via

intrathecal administration. Endotoxins are negatively charged

macromolecules as small as 20-30 kDa, varying in size due to bacterial origin,

the presence of divalent cations or biological detergents. Bacterial Endotoxin

is the significant pyrogen that has been identified as a contaminant in


82

parenteral products. The LAL reaction with Endotoxin requires pH neutrally and

optimum levels of divalent cations82. A uniform temperature of 37°C optimizes

the rate of reaction. Most Biotherapeutic drug products require dilution with

LAL Reagent Water (LRW) (J.F. Cooper, 1990) before testing to avoid

interference. Testing of serum, plasma, protein sample is subjected to

inhibition from serine protease inhibitors and this interference creates

problems in both Biotech and research fields58.

Materials and Methods:

Materials:

Limulus Amoebocyte Lysate(LAL), Control Standard Endotoxin(CSE), LAL

Reagent Water (LRW) were purchased from Endosafe U.S, 76,77Depyrogenated

10 X 75 mm assay tubes, 16X100mm dilution tubes and pyrogen free

Micropipette tips.

Principle of BET (Bacterial Endotoxins test):

The principle of Bacterial Endotoxin test is to detect presence of

Endotoxin in the given sample to which the test is applied using Lysate derived

from the animal Limulus Polyphemus. Limulus polyphemus is also called as

Horseshoe crab. It is found in the eastern cost of America. This animal is living

fossil as since last 150 million years there has been no physical or physiological

change taken place in the animal. The blood of the animal is blue in colour

due to the presence of Hemocyanin instead of Hemoglobin. The unique

property of the blood is to react with Lipopolysaccharide (LPS) present in the

membrane of Gram-negative bacteria.

Methods:

Procedure of LAL test:

Equal volume of test sample and LAL reagent is added in a

depyrogenated test tube of 10 X 75 mm and incubate this mixture at 37± 1°C

for 60±2 min. Then invert the tube by 180° and look for gel formation. If a gel

inside the test tube is able to maintain its integrity after inverting the tube to

180° then it is a positive reaction which indicates presence of Endotoxin in the

sample. Other than this any condition is considered as negative which


83

Indicates absence of Endotoxin in the sample (lesser than the lysate

sensitivity).

Endotoxin Limit:

Since Endotoxin is ubiquitous in nature there has to be some safety limit

to pass the product. The Endotoxin limit should be such that it will not cause

any harmful effects to patient. Appendix E of USFDA (United States Food and

Drug Administration) gives Endotoxin limits for various products. It also gives a

formula through which Endotoxin limit for new product can be calculated.

The formula is as follows.

Endotoxin Limit = K/ M (USP 32, 2009)

Where, ‘K’ is threshold pyrogenic dose in humans and animals. 5 EU/kg body

weight for parenteral drug and 0.2EU/kg body weight for intrathecal drug.

‘M’ Maximum dose administered to a patient per Kg body weight per hour

(Not heroic dose).

The limit formula for radiopharmaceuticals is:

175/V except for intrathecally-administered products.

14/V for intrathecal drugs.

‘V’ equals the maximum recommended dose, in ml, at the expiration date or

time. For drugs administered on a per Square Meter of Body Surface:

5 EU/ [(dose * 1.8 sq. m.)/ 70 Kg]

Product Testing:

For testing products equal volume of drug (sample) and LAL reagent is

taken and following tubes are prepared

Negative Product Control (NPC) - Sample + LAL

Positive Product Control (PPC) - Sample + CSE (2λ) + LAL

Negative Water Control (NWC) - LRW + LAL

Positive Water Control (PWC) - LRW + CSE (2λ) + LAL

Majority of times it has been a common observation that if a product is tested

directly it inhibits the LAL test and thus shows interference (J. van Noordwijk et

al., 1997).


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Interference:

Interference is defined as a significant difference between the end

points of positive water control and positive product control using standard

Endotoxin.

This interference could be either inhibition wherein the recovery of

Endotoxin is below than the expected or enhancement wherein the recovery

of Endotoxin is higher than expected.

Interference may occur due to following reasons:

Suboptimal pH:

The optimal pH for LAL reaction is in between about 6.8 to 7.4. If the

sample is too acidic or too basic it will inhibit enzymes involved in LAL reaction.

Hence it is very necessary to bring pH of reaction to neutral range. This can be

done using 1N Hcl or 1N NaOH for basic and acidic product respectively82.

Endotoxin modification:

Purified Endotoxin has tendency to form micelle formation which is due

to hydrophilic and hydrophobic interactions between LPS and water. This

aggregated Endotoxin escapes LAL test. Hence to avoid such problem vortex

mixing for samples is performed.

Container effects:

75Adsorption of Endotoxin on tube wall causes poor recovery of

Endotoxin in LAL test. So it is suggested to use high quality borosilicate glass

tubes because of its inert nature hence adsorption of Endotoxin is least.

Unbalanced cation levels:

Divalent cations play important role in LAL reactivity and dispersion of

Endotoxins. LAL test requires optimum concentration of Ca++and Mg++ ions. If

divalent cations are insufficient then Ca++and Mg++ ions are added externally.

Protein or enzyme modification:

Enzyme inactivation due to oxidants, proteolytic agents or specific

inactivators will cause inhibition.

Non-specific LAL activation:

Some molecules other than Endotoxin are known to react with LAL

reagent and give gel formation. This is enhancement reaction and is very

rare.


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Subjects:

Activated lymphocyte sample which is used for autologous immune

enhancement therapy for myeloid leukemia has been tested for sorting out of

interference problem.

90% of interference problems are solved by just diluting the sample. But how

much sample can be diluted so that it can still detect Endotoxin limit is given

by formula for Maximum Valid Dilution (MVD).

MVD = concentration of sample X Endotoxin Limit / Lysate sensitivity

Example :

Drug : Activated Lymphocytes

Endotoxin Limit : NMT 0.25 EU/mL

Lysate sensitivity : 0.03125 EU/mL

MVD = potency x E.L

λ

= 1mL/ mL X 0.25 EU/ml

0.03125EU/mL

MVD = 8

Product Validation:

Product needs to be validated before start for routine testing. Validation is

a test condition where an Endotoxin standard is detected with the same

efficiency in a test sample as it is in LRW. This validation study consists of two

different phases wherein in Phase I (Preliminary screening) involve interference

testing and Phase II consists of validation of product.

Significance of product validation is that it gives information on

whether there are any interfering factors in the drug product to the LAL test

and also it gives an idea of the approximate levels of Endotoxin content in the

drug product. It also covers manufacturing of product and formulation of the

product.

It is always advisable to carry out revalidation if product formulation is

changed and which is likely to affect the interference pattern of the product

for LAL test. Also revalidation is to be conducted for any product if there is any

change in manufacturing procedures or in vendor.


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Phase I: Preliminary Screening / interference Study

In this two identical series of product dilutions (two-fold dilutions), one spiked

with 2λ, and one left unspiked. The result of Phase I will tell you the non-

interfering dilution (NID) of the product, which is used for the actual validation

(Phase II). The non-interfering dilution (NID) is the first set of PPC that shows a

gel.

Example :

Drug : Activated Lymphocytes

Endotoxin Limit : NMT 0.25 EU/mg

Lysate sensitivity: 0.03125 EU/mL

MVD = potency x E.L

λ

= 1mL/mL X0.25 EU/ml

0.03125EU/mL

MVD = 8

Results and discussions: Sample Dilution 1:1 1:2 1:4 1:8

Unspiked -- -- -- --

Spiked -- -- -- --

Table: 1

This assay shows that there is inhibition up to 1:8 (MVD). Due to Inhibition LAL is

unable to detect the Endotoxins even in spiked sample After analyzing the

sample using different procedures, finally In order to sort out this inhibition

problem then the activated sample is heated at 55°C for 15min to coagulate

the proteins in the sample and this heat denaturation technique is applicable

to all protein samples. The denaturation won’t affect the Endotoxin because

Endotoxins can be denatured at 250°C for 30 minutes as per USP.

Assay results after Heat denaturation of the sample.

Sample Dilution 1:1 1:2 1:4 1:8

Unspiked -- ++ -- --

Spiked -- + + ++ ++

Table: 2


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This assay shows that there is inhibition upto 1:1 dilution and the spike recovery

at 1:2 dilutions onwards. Therefore the NID is 1: 2. It is advisable to validate the

product at not less than 1:4 dilution to take care of any batch to batch

variation during regular production. So 1: 4 dilution is chosen for product

validation.

Phase II: Validation of Product

For validation, test and compare two identical series of Endotoxin dilutions

bracketingλ; One prepared in LRW and another prepared in product diluted

to the proposed test dilution. Here dilution selected for validation is 1:4. (Hot

spike method).

Example of results:

Endotoxin/product

Replicates 0.0625EU/mL 0.03125EU/mL 0.015 EU/mL 0.007 EU/mL

1 + + - -

2 + + - -

3 + + - -

4 + + - -

Table: 3

Negative product control: --Geometric Mean = 0.03 EU/ml

Endotoxin/LRW

Replicates 0.0625EU/mL 0.03125EU/mL 0.015 EU/mL 0.007 EU/mL

1 + + - -

2 + + - -

3 + + - -

4 + + - -

Table: 4

Blank: -- Geometric Mean = 0.03 EU/ml

Successful validation requires that both series confirm label claim (Geometric

mean) within +/- one two-fold dilution. Validation is conducted at this dilution

on three batches of product.

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5.2 Lactobionic Acid

Abstract:

Lactobionic acid used in organ preservative during organ transplant. The

effect of varying the pH and ionic strength in Lactobionic acid was

investigated. The interfering factors inhibiting the Endotoxins in Lactobionic

acid while quantifying with Limulus Amoebocyte Lysate was sorted out by

neutralizing the ionic concentration in sample using alkali and validated the

test sample. Lactobionic acid is weak acid and having pH in the range 1 to 2.

Since it is a weak acid its ionic dissociation is very less. As we add strong alkali

like NaOH in to it, the pH rises immediately and after some time it comes down

to acidic range. After adjusting the pH the sample to be analyzed within an

hour to avoid interference.

Introduction:

Bacterial Endotoxin is one of the most potent activator of mammalian

immune system. In general, as per the United State Pharmacopeias (USP) the

threshold pyrogenic dose is 5 EU/kg/hr for parenteral drugs and 0.2 EU/kg/hr

for intrathecal drugs5. When Endotoxin enters into human blood these toxins

induces white blood cells (WBC) to release cytokines, such as tissue necrosis

factor (TNF), interleukin-1 and interleukin-8, which mediate a complex

biological response including pyrogensity, shock, coagulation and

inflammation5,4&7. Gram negative bacterial outer membrane

Lipopolysacchride (LPS) induces a cascade of defense mechanism that is

known as fever and inflammation2. So it is mandatory to check the presence

of Endotoxin level in Organ preservative before using it for preservation of

organs during transplant.

The LAL reaction with Endotoxin requires pH neutrality and optimum levels of

Na+ and divalent cations. A uniform temperature of 37°C optimizes the rate of

reaction. Most therapeutic drug products require dilution with LAL Reagent

Water (LRW) before testing to avoid interference, where inhibition is failure to

recover the positive control, and enhancement is excess recovery. There are

3 principle causes of invalid or inhibitory results in gel clot testing are 1. Loss of

purified Endotoxin used for product positive controls (PPC). 2. Adverse


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chemical conditions such as non-neutral pH or sub optimal levels of sodium

ions and divalent cations (Mg++ and Ca++). 3. Inadequate controlled test

parameters including testing accessories, reagents and analyst proficiency.

The aim of the study is to sort out the interfering factors which lead to the

diverse results in Lactobionic acid. The false positive results may cause severe

complication in the patients as discussed in literature.


Materials:

Lyophilized Limulus Amoebocyte Lysate of 0.0312 sensitivity (LAL), Control

Standard Endotoxin 5 Eu/ng (CSE), LAL Reagent Water (LRW) of Endosafe US,

Depyrogenated (250°C for 30 min) 10 X 75 mm assay tubes, 16X100 mm

dilution tubes, pyrogen free Micropipette tips, vortex mixture, 1N NaoH and

Lactobionic acid were used for determination of Endotoxin content by the gel

clot technique.

The sensitivity of the Lysate (labeled 0.0312 Eu/mL) was determined by using

known amount of E.coli Control Standard Endotoxin.

In the gel-clot techniques, the reaction end point is determined from dilutions

of the material under test in direct comparison with parallel dilutions or a

reference Endotoxin, and quantities of Endotoxins are expressed in Endotoxin

units.

1. Preparation of Standard stock solution and standard solutions: The CSE

having a defined potency of 50 EU/Vial was reconstituted with 5ml of LRW

and mixed intermittently for 30 minutes using a vortex mixture and this

concentrate was used to prepare 2λ, λ, λ/2 & λ/4, where λ is the labeled

claim sensitivity of Lysate.

2. Preparation of sample solution: Test samples were diluted to the required

concentrations based on the formulae MVD. MVD is the maximum valid

dilution, which is allowable dilution of the specimen at which the Endotoxin

limit can be determined. The general equation to determine MVD is

MVD = (Endotoxin limit X Concentration of sample solution)/ (λ). Where E.L is

the Endotoxin limit of the test sample, which is specified in the individual

monograph/ based on the E.L formula if not mentioned in the monograph, in

terms of volume or units of active drug (in EU/mg).


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3. Lactobionic acid sample preparation: Batch No: LBA-0109,

Potency=100mg/mL, E.L=0.005 Eu/mg, Lysate sensitivity is 0.0312 Eu/mL and

MVD = 16. The following test dilutions are prepared by 1:16 (6.25 mg/mL), 1:8

(12.5 mg/mL), 1:4 (25 mg/mL) & 1:2 (50 mg/mL). Lactobionic acid is weak acid

and having pH in the range 1 to 2.

Method:

Equal volume of test sample and LAL reagent is added in a depyrogenated

test tube of 10 X 75 mm and incubate this mixture at 37± 1°C for 60±2 min.

Then invert the tube by 180° and look for gel formation. If a gel inside the test

tube is able to maintain its integrity after inverting the tube to 180° then it is a

positive reaction which indicates presence of Endotoxin in the sample greater

than the limit. Other than this any condition is considered as negative which

indicates absence of Endotoxin in the sample (lesser than the lysate

sensitivity).

Product Testing:

For testing products equal volume of drug (sample) and LAL reagent is taken

and following tubes are prepared6.





Majority of times it has been a common observation that if a product is tested

directly it inhibits the LAL test and thus shows interference1&3.

Interference: Interference is defined as a significant difference between the

end points of positive water control and positive product control using

standard Endotoxin.

This interference could be either inhibition wherein the recovery of Endotoxin is

below than the expected or enhancement wherein the recovery of Endotoxin

is higher than expected.


91

Product Validation:

Product needs to be validated before start for routine testing. Validation is a

test condition where an Endotoxin standard is detected with the same

efficiency in a test sample as it is in LRW. This validation study consists of two

different phases wherein in Phase I (Preliminary screening) involve interference

testing and Phase II consists of validation of product.



and also it gives an idea of the approximate levels of Endotoxin content in the

drug product. It also covers manufacturing of product and formulation of the

product.





Phase I: Preliminary Screening / interference Study9

In this two identical series of product dilutions (two-fold dilutions), one

spiked with 2λ, and one left unspiked. The result of Phase I will tell you the non-

interfering dilution (NID) of the product, which is used for the actual validation

(Phase II). The non-interfering dilution (NID) is the first set of PPC that shows a

gel.

Results and discussions:

Lactobionic acid:


Unspiked -- -- -- --

Spiked -- -- -- --

Table: 1


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This assay shows that there is inhibition up to 1:16 (MVD) in Lactobionic acid.

Due to Inhibition LAL is unable to detect the Endotoxins even in spiked sample

After analyzing the sample using different procedures, finally In order to sort

out this inhibition problem the acidic pH of the Lactobionic acid (1-2) is

adjusted to 7-8 with 1N NaoH.

Lactobionic acid: (Results after adjusting the Acidic pH to the range of 7-8

with 1 N NaoH).


Unspiked ++ -- -- --

Spiked ++ ++ ++ ++

Table: 2

This assay shows no inhibition upto 1:2 dilution in Lactobionic acid and the

spike recovery at 1:2 dilutions onwards. Therefore the NID is 1:2 (Lactobionic

acid). It is advisable to validate the product at not less than MVD/4 to take

care of any batch to batch variation. So MVD/4 dilution is chosen for product

validation.

Phase II:

Validation of Product

For validation, test and compare two identical series of Endotoxin dilutions

bracketing λ; One prepared in LRW and another prepared in product diluted

to the proposed test dilution. Here dilution selected for validation is 1:4. (Hot

spike method).

Example of results:

Endotoxin/product

Replicates 0.0625 Eu/mL 0.0312 Eu/mL 0.0156 Eu/mL 0.0078 Eu/mL

1 + + - -

2 + + - -

3 + + - -

4 + + - -

Table: 3

Negative product control: --Geometric Mean = 0.0312 EU/ml


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Endotoxin/ LRW

Replicates 0.0625 Eu/mL 0.0312 Eu/mL 0.0156 Eu/mL 0.0078 Eu/mL

1 + + - -

2 + + - -

3 + + - -

4 + + - -

Table:4

Blank: -- Geometric Mean = 0.0312 EU/ml

Successful validation requires that both series confirm label claim (Geometric

mean) within +/- one two-fold dilution.

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5.3 Quantification of Bacterial Endotoxins in Medical devices:

Abstract: Like drug products, each lot of medical devices need to be

checked for bacterial Endotoxins for compliance before releasing into the

market. Endotoxins are found to be variable absorptive to the device surfaces

and leads to false negative results, to overcome this issue the desired test

device need to be filled with WFI/LRW and incubate it at 37°C for 30 min, so

that the Endotoxins stick to the walls of the medical device will be extracted

into the WFI/LRW. Then the WFI/LRW rinse will be tested and report the value

per ml of rinse. Then the reported value will be multiplies by total volume and

divide the same with the number of devices taken will gives us the Endotoxin

content in each medical device.


Materials:

Lyophilized Limulus Amoebocyte Lysate of 0.125 sensitivity (LAL), Control

Standard Endotoxin 5 Eu/ng (CSE), LAL Reagent Water (LRW) of Endosafe US,

Depyrogenated (250°C for 30 min) 10 X 75 mm assay tubes, 16X100 mm

dilution tubes, pyrogen free Micropipette tips, vortex mixture, Blood sets, I.V

Set, 1mL Syringe, 5mL Syringe and Sutures were used for determination of

Endotoxin content by the gel clot technique.

The sensitivity of the Lysate (labeled 0.125 Eu/mL) was determined by using

known amount of E.coli Control Standard Endotoxin.

In the gel-clot techniques, the reaction end point is determined from dilutions

of the material under test in direct comparison with parallel dilutions or a

reference Endotoxin, and quantities of Endotoxins are expressed in Endotoxin

units.

1. Preparation of Standard stock solution and standard solutions: The CSE

having a defined potency of 50 EU/Vial was reconstituted with 5ml of LRW

and mixed intermittently for 30 minutes using a vortex mixture and this

5.3 Quantification of Bacterial endotoxins in Medical devices

95

concentrate was used to prepare 2λ, λ, λ/2 & λ/4, where λ is the labeled

claim sensitivity of Lysate.

2. Preparation of sample solution: Test samples were diluted to the required

concentrations based on the formulae MVD. MVD is the maximum valid

dilution, which is allowable dilution of the specimen at which the Endotoxin

limit can be determined. The general equation to determine MVD is

MVD = (Endotoxin limit X Concentration of sample solution)/ (λ). Where E.L is

the Endotoxin limit of the test sample, specified for Medical devices.

3. Blood set rinse sample preparation: Potency 1 mL/mL , E.L= 0.73 Eu/mL ,

Lysate sensitivity is 0.125 Eu/mL and MVD = 96.The following test dilutions are

prepared by 1:1, 1:2, 1:3, 1:4 & 1:5.

4. I.V set rinse sample preparation: Potency 1 mL/mL, E.L= 1.25 Eu/mL , Lysate

sensitivity is 0.125 Eu/mL and MVD = 10. The following test dilutions are

prepared by 1:1, 1:2, 1:4, 1:8 & 1:10.

5. 1mL Syringe rinse sample preparation: Potency 1 mL/mL, E.L= 18.75 Eu/mL,

Lysate sensitivity is 0.125 Eu/mL and MVD = 150. The following test dilutions are

prepared by 1:9, 1:18, 1:37, 1:75 & 1:150.

6. 5mL Syringe rinse sample preparation: Potency 1 mL/mL, E.L= 3.75 Eu/mL,

Lysate sensitivity is 0.125 Eu/mL and MVD = 30. The following test dilutions are

prepared by 1:2, 1:4, 1:8, 1:16 & 1:30.

7. Sutures rinse sample preparation: Potency 1 mL/mL, E.L= 12 Eu/mL, Lysate

sensitivity is 0.125 Eu/mL and MVD = 30. The following test dilutions are

prepared by 1:6, 1:12, 1:24, 1:48 & 1:96.

Methods

Equal volume of test sample and LAL reagent is added in a

depyrogenated test tube of 10 X 75 mm and incubate this mixture at 37± 1°C

for 60±2 min. Then invert the tube by 180° and look for gel formation. If a gel


96

inside the test tube is able to maintain its integrity after inverting the tube to

180° then it is a positive reaction which indicates presence of Endotoxin in the

sample greater than the limit. Other than this any condition is considered as

negative which indicates absence of Endotoxin in the sample (lesser than the

lysate sensitivity).

Product Testing: For testing products equal volume of drug (sample) and LAL

reagent is taken and following tubes are prepared)





Product Validation: Product needs to be validated before start for routine

testing. Validation is a test condition where an Endotoxin standard is

detected with the same efficiency in a test sample as it is in LRW. This

validation study consists of two different phases wherein in Phase I (Preliminary

screening) involve interference testing and Phase II consists of validation of

product.



and also it gives an idea of the approximate levels of Endotoxin content in

the drug product. It also covers manufacturing of product and formulation of

the product.






97

(i) Blood Set:

Preliminary Screening Test

Product : Blood set

Concentration : 1 ml / mL

Endotoxin Limit : K X N V

K= 20 EU/Device

N = No of Devices

V = Volume of solution rinsed/ taken

E.L = 20 EU/Device x 3 Devices

82mL

= 0.73 EU/ml

MVD = Potency x E.L

Sensitivity of Lysate

= 1ml/ml x 0.73 EU/ml

0.125 EU/ml

= 5

Test Dilution : 1:1, 1:2, 1:3, 1:4 and 1: 5

Test sample Rinse: Three blood bags from each lot are taken and

completely filled with WFI and incubated it at 37°C for 30 min, Then the

Endotoxins stick to the walls of the bag are extracted into the WFI. Then the

WFI rinse was tested and reported the value per ml of rinse. Then the

reported value was multiplied by total volume and divided the same with

the number of devices taken, it gives us the Endotoxin content in each

Blood bag.

Test Dilutions NPC PPC

1:1 − − + +

1:2 − − + +

1:3 − − + +

1:4 − − + +

1:5 − − + +

++: Gel Formation − − : No Gel Formation


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Conclusion: Carried out screening and observed Non Interfering Dilution (NID)

at MVD/8 (1:1). Non Interfering dilution is 1ml/ mL. Endotoxins

content is < 4 EU/ device.

End-Product Endotoxins Test79:

Product : Blood set

Preparation : Product Concentration : 1mL / mL

Endotoxin Limit : 20 EU/Device

MVD : 5

Test Dilution : 1:2

Results : Test Endotoxin

Concentration EU/mL.

NEG

Control

NEG

Product

Control

Test

End

point.

Log of

End

point.

Geometric Mean

=A log (log of End

point.)

Material 2λ λ ½λ ¼λ EU/mL

+ + - − − 0.125 -0.9030

+ + - − − 0.125 -0.9030

+ + - − 0.125 -0.9030

CSE

Water

Control

+ + - − 0.125 -0.9030

+ + - − − 0.0625 -1.2041

+ + - − − 0.0625 -1.2041

+ + - − − 0.125 -0.9030

Positive

Product

Control

+ + - − − 0.125 -0.9030

Antilog

(-3.612/4)

=Antilog –0.903

=0.125EU/mL

Antilog

(-4.2142/4)

=Antilog–1.05355

=0.088EU/mL

Interpretation :

Test results are : Valid

Comments : Product validation for Blood set was carried out at MVD/8 (1:2).

The GM of the end points in Endotoxin / LRW was 0.125EU/mL and

Endotoxin/product was 0.088 EU/mL which is in between 2λ and ½ λ, which

indicates no inhibition and enhancement at test dilution 1:2.


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(II) I.V SET


Product : I.V Set


Endotoxin Limit : K X N

V

K= 20 EU/Device

N = No of Devices



48mL

= 1.25 EU/ml

MVD = Potency x E.L



0.125 EU/ml

= 10


Test sample Rinse: Three I.V Sets from each lot are taken and completely

filled with WFI and incubated it at 37°C for 30 min, Then the Endotoxins stick

to the walls of the bag was extracted into the WFI. Then the WFI rinse was

tested and reported the value per ml of rinse. Then the reported value was

multiplied by total volume and divide the same with the number of devices

taken, it gives us the Endotoxin content in each I.V bag.


1:1 − − − −

1:2 − − + +

1:4 − − + +

1:8 − − + +

1:10 − − + +



100

Conclusion: Carried out screening and observed Non Interfering Dilution (NID)

at 1:2 dilution, at 1:1 the rinse of the I.V set is showing inhibition.

Non Interfering dilution is 1:2 dilution. Endotoxins content is < 2.5

EU/ device.

End-Product Endotoxins Test:

Product : I.V set



MVD : 10

Test Dilution : 1:2



NEG

Control

NEG

Product

Control

Test

End

point.

Log of

End

point.

Geometric Mean

=A log (log of End

point.)


+ + - − − 0.125 -0.9030

+ + - − − 0.125 -0.9030

+ + - − 0.125 -0.9030

CSE

Water

Control

+ + - − 0.125 -0.9030

+ + - − − 0.0625 -1.2041

+ + - − − 0.0625 -1.2041

+ + - − − 0.125 -0.9030

Positive

Product

Control

+ + - − − 0.125 -0.9030

Antilog

(-3.612/4)

=Antilog –0.903

=0.125EU/mL

Antilog

(-4.2142/4)

=Antilog–1.05355

=0.088EU/mL

Interpretation :


Comments : Product validation for I.V set was carried out at 1:4 dilution. The

GM of the end points in Endotoxin / LRW was 0.125EU/mL and


indicates no inhibition and enhancement at test dilution 1:4 dilution.


101

(iii) 1 mL SYRINGE


Product : 1 ml Syringe



V

K = 20 EU/Device

N = No of Devices



3.2 mL

= 18.75 EU/ml

MVD = Potency x E.L



0.125 EU/ml

= 150


Test sample Rinse: Three 1 ml Syringes from each lot are taken and


Endotoxins stick to the walls of the Syringe was extracted into the WFI. Then

the WFI rinse was tested and report the value per ml of rinse. Then the

reported value was multiplied by total volume and divide the same with the

number of devices taken, it gives us the Endotoxin content in each 1 ml

Syringe.


1:9 − − + +

1:18 − − + +

1:37 − − + +

1:75 − − + +

1:150 − − + +



102

Conclusion: Carried out screening and observed Non Interfering Dilution

(NID) at 1:9 dilutions. Non Interfering dilution is 1:9 dilution.

Endotoxins content is < 1.5 EU/ device.





MVD : 150

Test Dilution : 1:18

Results :

Test Endotoxin


NEG

Control

NEG

Product

Control

Test

End

point.

Log of

End

point.

Geometric Mean

=A log (log of End

point.)


+ + - − − 0.125 -0.9030

+ + - − − 0.125 -0.9030

+ + - − 0.125 -0.9030

CSE

Water

Control

+ + - − 0.125 -0.9030

+ + - − − 0.0625 -1.2041

+ + - − − 0.0625 -1.2041

+ + - − − 0.125 -0.9030

Positive

Product

Control

+ + - − − 0.125 -0.9030

Antilog

(-3.612/4)

=Antilog –0.903

=0.125EU/mL

Antilog

(-4.2142/4)

=Antilog–1.05355

=0.088EU/mL

Interpretation :


Comments : Product validation for 1 ml Syringe was carried out at 1:8

dilution. The GM of the end points in Endotoxin / LRW was 0.125EU/mL and




103

(iv) 5 mL SYRINGE





V

K = 20 EU/Device

N = No of Devices



16 mL

= 3.75 EU/ml

MVD = Potency x E.L



0.125 EU/ml

= 30


Test sample Rinse: Three 5 ml Syringes from each lot are taken and


Endotoxins stick to the walls of the Syringe was extracted into the WFI. Then

the WFI rinse was tested and report the value per ml of rinse. Then the

reported value was multiplied by total volume and divide the same with the

number of devices taken, it gives us the Endotoxin content in each 5 ml

Syringe.


1:2 + + + +

1:4 − − + +

1:8 − − + +

1:16 − − + +

1:30 − − + +



104


(NID) at MVD/8 (1:2) dilution. Non Interfering dilution is 1:2

dilution. Endotoxins content is < 1.6 EU/ device.





MVD : 30

Test Dilution : 1:4

Results :

Test Endotoxin


NEG

Control

NEG

Product

Control

Test

End

point.

Log of

End

point.

Geometric Mean

=A log (log of End

point.)


+ + - − − 0.125 -0.9030

+ + - − − 0.125 -0.9030

+ + - − 0.125 -0.9030

CSE

Water

Control

+ + - − 0.125 -0.9030

+ + - − − 0.0625 -1.2041

+ + - − − 0.0625 -1.2041

+ + - − − 0.125 -0.9030

Positive

Product

Control

+ + - − − 0.125 -0.9030

Antilog

(-3.612/4)

=Antilog –0.903

=0.125EU/mL

Antilog

(-4.2142/4)

=Antilog–1.05355

=0.088EU/mL

Interpretation :


Comments : Product validation for 5ml Syringe was carried out at MVD/4

(1:4). The GM of the end points in Endotoxin / LRW was 0.125EU/mL and




105

(V) SUTURES


Product : Suture



V

K= 20 EU/Device

N = No of Devices



5mL

= 12 EU/ml

MVD = Potency x E.L


= 1ml/ml x 12 EU/ml

0.125 EU/ml

= 96


Test sample Rinse: Three Sutures sets from each lot are taken and soaked

them in 5 ml WFI and incubated it at 37°C for 30 min, and then the

Endotoxins stick to the suture roll was extracted into the WFI. Then the WFI

rinse was tested and report the value per ml of rinse. Then the reported

value was multiplied by total volume and divide the same with the number

of devices (Sutures), it gives us the Endotoxin content in each Suture roll.


1:6 − − + +

1:12 − − + +

1:24 − − + +

1:48 − − + +

1:96 − − + +



106


(NID) at MVD/16 (1:1). Non Interfering dilution is 1:6 (I Part of

Rinse and 5 parts of LRW) Endotoxins content is < 1.25 EU/

device (Per suture roll).


Product : Suture



MVD : 96

Test Dilution : 1:12



NEG

Control

NEG

Product

Control

Test

End

point.

Log of

End

point.

Geometric Mean

=A log (log of End

point.)


+ + - − − 0.125 -0.9030

+ + - − − 0.125 -0.9030

+ + - − 0.125 -0.9030

CSE

Water

Control

+ + - − 0.125 -0.9030

+ + - − − 0.0625 -1.2041

+ + - − − 0.0625 -1.2041

+ + - − − 0.125 -0.9030

Positive

Product

Control

+ + - − − 0.125 -0.9030

Antilog

(-3.612/4)

=Antilog –0.903

=0.125EU/mL

Antilog

(-4.2142/4)

=Antilog–1.05355

=0.088EU/mL

Interpretation :


Comments : Product validation for Suture (roll) was carried out at MVD/8

(1:12). The GM of the end points in Endotoxin / LRW was 0.125EU/mL and


indicates no inhibition and enhancement at test dilution MVD / 8 (1:12).

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